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2 edition of Site-specific recombination of the 2-micron plasmid of yeast. found in the catalog.

Site-specific recombination of the 2-micron plasmid of yeast.

Brenda Jean Andrews

Site-specific recombination of the 2-micron plasmid of yeast.

by Brenda Jean Andrews

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Published .
Written in English


The Physical Object
Pagination[184] leaves
Number of Pages184
ID Numbers
Open LibraryOL16350767M

One interesting yeast plasmid is called the 2u circle. The 2u circle is a kb circular, extrachromosomal element found in the nucleus of most Saccharomyces cerevisiae strains. The 2u circle doesn't give cells that carry it any apparent selective advantage, but it is stably maintained at about 50 to copies per haploid genome of the yeast. Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors can also be controlled if specific regulatable elements are included (reviewed here).

  In the case of unequal partitioning of 2μ plasmid clusters during cell division, a recombination‐based plasmid amplification system can restore high copy numbers. The 2μ plasmid codes for a site‐specific recombinase (FLP1) and bears two corresponding recognition sites (FRT sites) (Broach and Hicks, ).   Overview. This is a protocol for inserting a piece of DNA into a plasmid using homologous recombination in yeast. It works roughly as follows: a plasmid (CEN or 2-micron with selectable yeast and bacterial markers) is digested with a restriction enzyme somewhere in the middle of where you would like the DNA to insert into the plasmid.

The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of per haploid cell. The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. Site-specific recombination and partitioning systems in the stable high copy propagation of the 2-micron yeast plasmid. Prog Nucleic Acid Re. 72 Johnson. LJ.


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Site-specific recombination of the 2-micron plasmid of yeast by Brenda Jean Andrews Download PDF EPUB FB2

Abstract Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two base-pair precise inverted repeats across which a site-specific inversion event occurs in vivo. This inversion is promoted by a plasmid-encoded function called "FLP."Cited by: Broach, J.R., V.R.

Guarascio, and M. Jayaram () Recombination within the yeast plasmid 2-micron circle is site-specific. Cell – PubMed CrossRef Google Scholar by: 4.

An impressive application of Flp in eukaryotes has been the creation of mosaic flies in Drosophila by site-specific recombination between homologous chromosomes.

Aside from its natural role in the physiology of the 2μm plasmid, Flp has been used to perform many artificial functions in its native host Saccharomyces by: Abstract Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two base-pair precise.

Abstract. Most strains of the yeast Saccharomyces cerevisiae harbor several copies of a 2-micron plasmid circle DNA termed "2 micron." This circular plasmid contains two base-pair precise inverted repeats across which a site-specific inversion event occurs in by:   The 2-micron plasmid of the yeast Saccharomyces cerevisiae encodes a site-specific recombinase (FLP) that promotes inversion across a unique site contained in each of the base-pair inverted repeats of the plasmid.

We have studied the topological changes generated in supercoiled substrates after exposure to the purified FLP protein in vitro.

The yeast 2-micron plasmid is a 6,bp circular DNA molecule occurring in many yeast strains. It is generally present at 50 to copies per cell and has been sequenced in its entirety (11). The plasmid encodes at least four protein products (Ref.

6; Volkert et al., this Volume), which are involved primarily in plasmid maintenance. Abstract The site-specific recombination system (FLP) encoded by the yeast plasmid 2-micron circle can also act in yeast on the inverted repeats of the bacterial transposon Tn5.

The efficiency of this recombination is dependent on the location of Tn5 within the 2-micron circle genome but can be as high as that observed for 2-micron circle itself.

McLeod, M., F. Volkert, and J. Broach () Components of the site-specific recombination system encoded by the yeast plasmid 2-micron circle. Cold Spring Harbor Symp. Quant. Biol. – PubMed CrossRef Google Scholar. The 2 micron plasmid of Saccharomyces cerevisiae is a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40–60 per haploid cell.

The plasmid is able to persist in host populations with almost chromosome-like stability with the help of a partitioning system and a copy number control system. Site-specific recombination provides an efficient complementary set of methods for further manipulat- ing the transgene.

In contrast to homologous recom- bination, site-specific recombination relies not on the endogenous recombinational machinery of the cell, but on an exogenously supplied recombinase. Broach JR, Guarascio VR, Jayaram M.

Recombination within the yeast plasmid 2mu circle is site-specific. Cell. May; 29 (1)– Futcher AB. Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae.

J Theor Biol. Mar. Most strains of the yeast, Saccharomyces cerevisiae, harbor about copies of an autonomously replicating plasmid, the 2-μm gh the plasmid confers no advantage to its host, it is very stable. Because of its high copy-number and stability, the 2-μm plasmid has been a useful model for studying DNA replication, recombination, regulation of gene expression, and plasmid.

The R gene product (R protein) of Zygosaccharomyces rouxii plasmid pSR1 catalyzes site-specific recombination within a 58 base-pair (bp) sequence present in the bp inverted repeats of this plasmid. The R protein was produced in Escherichia coli and partially purified.

The partially purified protein catalyzed site-specific recombination in vitro without the supply of an energy source. Veit BE, Fangman WL.

Copy number and partition of the Saccharomyces cerevisiae 2 micron plasmid controlled by transcription regulators. Mol Cell Biol. Nov; 8 (11)– [Europe PMC free article] [Google Scholar] Volkert FC, Broach JR.

Site-specific recombination promotes plasmid amplification in yeast. Cell. Yeast episomal plasmids (YEps) are shuttle vectors. They can replicate in E.

coli and also in yeast. The yeast episomal plasmids and has the following parts: 1. The 2 Micron (µm) Plasmid, 2. LEU2 Gene, and 3. The pBR Part # 1. The 2 Micron (µm) Plasmid: This plasmid is found in several strains of yeast, Saccharomyces cerevisiae. 2-micron circle inheritance is affected by ARS-plasmids.

During the course of previously published experiments [], we observed that yeast transformed with ARS-plasmids were cured of 2-micron plasmids with a higher than expected frequency (data not shown).The majority of laboratory yeast strains, including the Wderived strains we use, contain the naturally occurring 2-micron plasmid.

The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene.

replicating plasmid called 2-micron circle DNA. This plasmid codes for a site-specific recombinase, the FLP protein which promotes recombination across two base pair inverted repeats of the plasmid DNA.

We have cloned the FLP gene under the control of a strong Escherichia coli promoter and have hyperproduced the. Broach JR, Guarascio VR, Jayaram M () Recombination within the yeast plasmid 2µ circle is site-specific.

Cell – doi: /(82) PubMed CrossRef Google Scholar Cairns BR, Lorch Y, Li Y et al () RSC, an essential, abundant chromatin-remodeling complex. We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila.

Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The 2 micron plasmid of Saccharomyces cerevisiae codes for a site-specific recombinase, the FLP protein, that catalyzes efficient recombination across two base-pair (bp) inverted repeats of the plasmid DNA both in vivo and in vitro.Tyrosine family site-specific recombinases (YRs), named after the active site tyrosine nucleophile they utilize for DNA strand breakage, are widely distributed among prokaryotes.

They were thought to be nearly absent among eukaryotes, the budding yeast lineage (Saccharomycetaceae) being an exception in that a subset of its members houses nuclear plasmids that code for YRs (1, 2).